Our overarching goal is to develop a dual photoacoustic-ultrasound guided drug delivery for cancer. Improvement of therapeutic delivery to solid tumors is urgently needed to address toxicity of the systemic dosing of cancer drugs. Development of efficient delivery methods has been slow; overall long-term survival gains of cancer patients over the past decades are very modest. We propose a fluorocarbon-particle-modified red blood cell (RBC) as acoustically active drug carrier. These natural particles offer excellent drug loading capacity, and can be easily used for site-specific targeting, by decorating them with ligands towards tumor vasculature biomarkers or magnetic particles. Decorating RBCs with perfluorocarbon nanodroplets allows rapid rupture of RBC membrane in response to a single short ultrasound pulse, resulting in rapid (<1 sec) payload release. By combining these carriers with ultrasound and photoacoustic imaging or SPECT, we expect that ultrasound-controlled release of encapsulated drugs, guided by imaging, will bring significant antitumor effects. We have demonstrated ultrasound-mediated RBC rupture and dye release of entrapped in vitro. We will then encapsulate in aaRBCs a chemotherapeutic doxorubicin and immunotherapeutic imiquimod (currently used topically) and demonstrate delivery in an in vivo murine tumor model. Aim 1. Elucidate ultrasound triggered release mechanism of red blood cell carriers. Acoustically active red blood cells (aaRBCs) are made by attaching perfluorocarbon nanodroplets (NDs) to the RBC membrane. Drug release is triggered by acoustic activation, causing the NDs to vaporize into micron-size microbubbles and rupture the RBC membrane. Our high-speed camera, used to capture ultra-fast dynamics of droplets and microbubbles, will be used to image vaporization of droplets attached to RBC membranes. Optimal aaRBC and ultrasound design parameters that trigger release of >50% of encapsulated drug will be determined in vitro. Aim 2. Achieve targeting and imaging functionality of long-circulating aaRBCs. aaRBC molecular targeting to the vascular endothelium will be achieved through conjugation of a cyclic Arg-Gly-Asp (RGD) peptide and vascular endothelial growth factor (scVEGF) to the RBC membrane. Magnetic targeting will be achieved by loading magnetic nanoparticles. Radiolabeled aaRBCs will be used to assess biodistribution of targeted aaRBCs in a murine tumor model, augmented with SPECT imaging. An approved NIR dye, indocyanine green (ICG), will be encapsulated into RBCs and imaged using a photoacoustic system, for real-time release control. Aim 3. Demonstrate image-guided therapy in a mouse tumor model with aaRBCs and ultrasound. We will use a targeted aaRBC loaded with doxorubicin and/or imiquimod, along with ICG dye. We will trigger release and monitor delivery using a photoacoustic/ultrasound and SPECT. Therapy will be performed in a mouse hindlimb MC38 colon adenocarcinoma; delay of tumor growth assessed post-treatment. Localized delivery of encapsulated agents will increase therapeutic index and minimize the drug dose and off-target drug deposition.